It has been the practice for 50 years or more to transfuse whole blood, and more recently blood components, from one or more donors to other persons. With the passage of time and accumulation of research and clinical data, transfusion practices have improved greatly. One aspect of current practice is that whole blood is rarely administered; rather, patients needing red blood cells are given packed red cells (hereinafter PRC), and patients needing platelets are given platelet concentrate. These components are separated from whole blood by centrifuging, the process providing, as a third product, plasma, from which various other useful components are obtained. In addition to the three above-listed components, whole blood contains white blood cells (known collectively as leucocytes) of various types, of which the most important are granulocytes and lymphocytes. White blood cells provide protection against bacterial and vital infection.
In the mid to late seventies, a number of investigators proposed that granulocytes be separated from donated blood and transfused into patients who lacked them, for example, those whose own cells had been overwhelmed by an infection. In the resulting investigations, it became apparent that this practice is generally harmful, since patients receiving such transfusion developed high fevers, had other adverse reactions, and often rejected the transfused cells. Further, the transfusion of packed cells or whole blood containing donor leucocytes can be harmful to the recipient in other ways. Some of the viral diseases induced by transfusion therapy, e.g., Cytomegaloviral Inclusion Disease, which is a life threatening infection to newborns and debilitated adults, are transmitted by the infusion of homologous leucocytes. Another life-threatening phenomenon affecting immunocompromised patients is Graft versus host disease (GVH); a disease in which the transfused leucocytes actually cause irreversible damage to the blood recipient's organs including the skin, gastrointestinal tract and neurological system. More recently, retroviruses such as HIV (AIDS) and HTLV1 have become a threat. Since some viruses, including several of those described above, are resident in the leucocytes, the removal of leucocytes is regarded as beneficial.
Conventional red cell transfusions have also been indicted as adversely influencing the survival of patients undergoing surgery for malignancy of the large intestine. It is believed that this adverse effect is mediated by the transfusion of agents other than donor red blood cells, including the donor's leucocytes.
Removal of leucocytes to sufficiently low levels to prevent the undesired reactions, particularly in packed red cells which have been derived from freshly drawn blood, is an objective of this invention.
In the currently used centrifugal methods for separating blood into the three basic fractions (packed red cells, platelet concentrate, and plasma), the leucocytes are present in substantial quantities in both the packed red cells and platelet concentrate fractions. It is now generally accepted that it would be highly desirable to reduce the leucocyte concentration of these blood components to as low a level as possible. While there is no firm criterion, it is generally accepted that many of the undesirable effects of transfusion would be reduced if the leucocyte content were reduced by a factor of about 100 or more prior to administration to the patient. This approximates reducing the total content of leucocytes in a single unit of PRC (the quantity of PRC obtained from a single blood donation) to less than about 1.times.10.sup.7. Recently it has become more widely perceived that in order to prevent viral infection by transfused blood, factors of reduction should be more than 100, preferably more than 1000, and more preferably 30,000 or 100,000 fold or more, such as 1,000,000 fold.
One of the most effective means of reducing leucocyte content that has been discovered hitherto is disclosed in U.S. Pat. No. 4,925,572 (application Ser. No. 07/259,773, filed Oct. 19, 1988), which is directed towards the bedside filtration of PRC. By contrast, this invention relates to the filtration of freshly drawn whole blood and of fresh PRC, that is, PRC that is filtered within 24 hours, and more preferably within 6 hours, of the time the blood was drawn. The behavior of fresh PRC is very different from that of the 2 to 35 day old blood that is described in U.S. Pat. No. 4,925,572. The standards for leucocyte depletion are also very different; the above copending application has as its objective leucocyte reduction by a factor of up to about 3000 to 10,000 and, while this is excellent for many purposes, the objective of the present application is leucocyte reduction by a factor in excess of about 30,000, and preferably of about 1,000,000 or more.
Defining a Unit of Blood and a Unit of Packed Red Cells
Blood banks in the United States commonly draw about 450 milliliters (ml) of blood from the donor into a bag which usually contains an anticoagulant to prevent the blood from clotting. Herein the quantity drawn during such a donation is defined as a unit of whole blood.
While whole blood is to a degree used as such, most units are processed individually by centrifugation to produce one unit of PRC. The volume of a unit of PRC varies considerably dependent on the hematocrit (percent by volume of red cells) of the drawn blood, which is usually in the range of about 37% to about 54%; and the hematocrit of the PRC, which varies over the range from about 50 to over about 80%, depending on whether yield of one or another blood compound is to be minimized. Most PRC units are in the range of about 170 to about 350 ml, but variation below and above these figures is not uncommon.
Drawn whole blood may alternatively be processed by separating the red cells from the plasma, and resuspending them in a physiological solution. A number of physiological solutions are in use. The red cells so processed may be stored for a longer period before use, and with some patients there may be some advantages in the removal of plasma. "Adsol" is the trade name of one such procedure, and SAG-M is a variant used in parts of Europe.
As used herein the term "fresh blood product" includes anti-coagulated whole blood, packed red cells obtained therefrom, and red cells separated from plasma and resuspended in physiological fluid, in all cases processed including filtration within about 24 hours and preferably within 6 hours of when the blood was drawn.
In parts of the world other than the United States, blood banks and hospitals may draw less or more than about 450 ml of blood; herein, however, a "unit" is always defined by the United States' practice, and a unit of PRC or of red cells in physiological fluid is the quantity derived from one unit of whole blood.
As used herein, PRC refers to the blood products described above, and to similar blood products obtained by other means and with similar properties.
Previously Available Means to Remove Leucocytes from PRC
The Spin-Filter system for obtaining leucocyte depleted packed red cells is described by Parravicini, Rebulla, Apuzzo, Wenz and Sirchia in Transfusion 1984; 24:508-510, and is compared with other methods by Wenz in CRC Critical Reviews in Clinical Laboratory Sciences 1986; 24:1-20. This method is convenient and relatively inexpensive to perform; it has been and continues to be used extensively. However, the efficiency of leucocyte removal, while generally about 90% or better, is not sufficiently high to prevent adverse reactions in some patients.
Centrifugation methods are available which produce lower levels of leucocytes in red cells, but these are laboratory procedures which are very costly to operate, and sterility of the product is compromised to a degree such that it must be used within 24 hours.
Other methods for leucocyte depletion, such as saline washing or deglycerolizing frozen red cells, have been or are used, but these have disadvantages for economical, high reliability service.
A number of devices have been proposed in which fibers are packed into housings, and whole blood passed through them in order to remove microaggregates and a portion of the leucocyte content. These devices have, when reduced to practice, all required saline to be applied either before or after use, or both before and after use, and are very poorly suited for blood bank use.
Characteristics Desirable in a Leucocyte Depletion Device
An ideal device for leucocyte depletion intended for use by blood banks would be inexpensive, relatively small, and be capable of processing one unit of PRC rapidly, for example in less than about one hour, and reduce the leucocyte content to the lowest possible level. Because of the high cost and limited availability of red blood cells, this ideal device would deliver the highest possible proportion of the red cells present in the donated blood. Such a device is an object of this invention.
Devices which have previously been developed in attempts to meet this objective have been based on the use of packed fibers, and have generally been referred to as filters. However, it would appear on preliminary review that processes utilizing filtration based on separation by size cannot succeed for two reasons. First, the various types of leucocytes range from granulocytes and macrocytes, which can be larger than about 15 .mu.m, to lymphocytes, which are in the 5 to 7 .mu.m range. Together, granulocytes and lymphocytes represent the major proportion of all of the leucocytes in normal blood. Red blood cells are about 7 .mu.m in diameter, i.e., in size they are in the range of one of the two major components which must be removed. Secondly, all of these cells deform so as to pass through much smaller openings than their normal size. Accordingly, and because it is readily observed by microscopic examination that leucocytes are adsorbed on a variety of surfaces, it has been widely accepted that removal of leucocytes is accomplished mainly by adsorption, rather than by filtration. An unexpected and surprising result of this invention, however, is that filtration through certain filters having a controlled pore size is critical to reach the target levels of leucocyte depletion.
Blood Component Recovery
In the preceding section, reference was made to the desirability of recovering a high proportion of the red cells delivered to the separation device. There are several causes for reduced recovery of red cells:
(a) Losses due to hold up within the connecting tubing; PA1 (b) Losses due to liquid which remains within the device itself at the conclusion of the filtration; PA1 (c) Losses due to adsorption on the surfaces of the device, or due to mechanical entrapment within the device; PA1 (d) Loss due to clogging of the filter prior to completion of the passage of the full unit of blood; and PA1 (e) Losses due to contact with incompatible surfaces, which can cause clotting. PA1 1. The volume of 1 gram of fibrous aggregate=1/D PA1 2. The volume of 1 gram of fibers=1/d PA1 3. The voids volume, V, is the total aggregate volume less the fiber volume or 1/D-1/d PA1 (a) An important aspect of this invention is the discovery that fibrous media treated to convert the fiber surfaces to a particular range of CWST perform better with respect to priming time, leucocyte depletion efficiency, and resistance to clogging than do fibrous media with CWST values outside of those ranges. PA1 (b) Synthetic fiber media whose CWST values have been elevated by grafting have, when hot compressed, superior fiber-to-fiber bonding and are for this reason preferred for use in making the preformed elements used in this invention. PA1 (c) Detrimental effects such as occasional clotting of blood associated with non-wetting as described in previous sections are avoided. PA1 (d) Devices made using unmodified synthetic fibers are recommended to be flushed with saline prior to use. This operation is undesirable since it causes blood loss due to hold-up within the complex tubing arrangement required, adds to cost, operation time, and operation complexity, and increases the probability that sterility may be lost. The need for preflushing is obviated by raising the CWST to the values disclosed in this invention. PA1 (a) Previously disclosed devices have used a relatively small cross sectional area perpendicular to the flow path, and are correspondingly longer with respect to the depth of their flow paths. The preferred devices in accordance with this invention are larger in cross sectional area perpendicular to the flow path and correspondingly shorter in depth of flow. This improvement in design helps to prevent clogging by PRC containing unusually high quantities of gels or microaggregates. PA1 (b) In order to make the larger cross sectional area economic and practical and to obtain the required degree of prefiltration, the filter components used in accordance with this invention are preferably preformed prior to assembly to closely controlled dimension and density parameters so as to form, in whole or in part, integral elements, self-contained and independent of other elements until assembled into a device in accordance with the subject invention. By "integral element" is meant a unitary, complete structure having its own integrity and, as mentioned, self-contained and independent of the other integral elements until assembled. PA1 (c) While it might be thought that freshly drawn blood would be free of aggregates and gels, hence prefiltration would not be required to prevent filter clogging, it has been the experience of the applicants that freshly drawn blood does occasionally clog a filter capable of producing a filtrate with less than about 10.sup.4 leucocytes per unit of PRC, corresponding to a reduction in leucocyte content by a factor of about 10.sup.5. PA1 (d) While the gel prefilter is extremely efficient in removing gels with a very small increase in pressure drop, and frequently removes as well quantities of microaggregates suspended in the gels, it removes only a portion of any microaggregates that may be present. Removal of the smaller microaggregates may be accomplished by one, two, or more layers of prefiltration using filter media of intermediate pore diameter which may either be separate preformed layers, but which in a preferred form of this invention are integral with part or all of the adsorption/filtration element. PA1 (e) The housing into which the element assembly is sealed is uniquely designed to achieve convenience of use, rapid priming, and efficient air clearance, this last leading to further reduction in hold-up of PRC. PA1 (f) The lateral dimensions of the elements are larger than the corresponding inner dimensions of the housing into which they are assembled. For example, if the elements are of disc form, the disc outside diameter is made about 0.1 to about 0.5% larger than the housing inside diameter. This provides very effective sealing by forming an interference fit with no loss of effective area of the elements, and contributes further towards minimization of the blood hold-up volume of the assembly.
Capacity
As separated from whole blood in current blood banking practice, packed red cells contain not only a proportion of the leucocytes present in the blood as drawn from the donor, but also some platelets (which tend to be very adhesive), fibrinogen, fibrin strands, tiny fat globules, and numerous other components normally present in small proportions. Also contained are factors added at the time the blood is drawn to prevent clotting, and nutrients which help to preserve the red cells during storage.
During the centrifuging process which concentrates the red cells and partially separates them from the remaining components there is a tendency for microaggregates to form in PRC. These may comprise some red cells together with leucocytes, platelets, fibrinogen, fibrin, fat, and other components. Gels, which may be formed by fibrinogen and/or fibrin, may also be present in PRC produced by blood banks.
If the leucocyte depletion device comprises a porous structure, microaggregates, gels and occasionally fat globules tend to collect on or within the pores, causing blockage which inhibits flow.
Ease and Rapidity of Priming
Ease of use is an important characteristic of any leucocyte depletion system. As noted above, for leucocyte depletion devices, ease of priming is a particularly important factor. The term "priming time" refers to start-up of flow of PRC from the bag through the filter to the patient, and is the time required to fill the filter housing from its inlet to its outlet. An object of this invention is to maintain a short priming time, preferably less than about 30 to about 120 seconds, to conserve technician time.
Preconditioning of Leucocyte Depletion Devices Prior to Priming
A number of devices in current use require pretreatment prior to passing blood, usually consisting of passing physiological saline. The necessity for such an operation is very undesirable in blood bank processing because it complicates the procedure, requires technician time, and puts maintenance of sterility at risk.
The reasons for using such pretreatment vary. They include removal of acid hydrolysate developed during steam sterilization of devices containing cellulose acetate fibers, assurance of freedom from foreign solids which may be present in natural fibers, and if the fibers are hygroscopic to prevent hemolysis (loss of the integrity of red blood cells with subsequent loss of their contents to the external milieu).
An objective of this invention is a leucocyte depletion device which requires no preconditioning prior to processing PRC derived from freshly drawn blood.
Definition of Voids Volumes
The concept of "voids volume" is related to, but distinguishable from, the term "bulk density". In fact, the term bulk density is misleading when referring to a broad spectrum of fibers with large variations in specific gravity. For example, polyester fibers may have a specific gravity of about 1.38 while inorganic fibers prepared from zirconia may have a specific gravity of greater than 5. Thus, in carrying out the instant invention, references to voids volume should not be confused with the term bulk density.
The concept of voids volume may be explained as follows.
Calculation of Voids Volume, Given Bulk Density and Fiber Density
Bulk density, D, is the weight of a given volume of fibrous aggregate divided by its apparent volume. Normally this is expressed in g/cc.
By fibrous aggregate is meant one or more fibers occupying a given or apparent volume, e.g., a mass of non-woven intertangled fibers with a certain proportion of voids or spaces within the mass.
In order to calculate the voids volume, V, the density, d, of the fibers must be known. The density, d, is also expressed in g/cc.
Example: ##EQU1##
The following table illustrates the difference between specifying voids volume and density. As illustrated there, at constant density, a column of glass fibers (glass being much more dense than, e.g., polypropylene) has a voids volume of 94% versus only 83.3% for a column of polypropylene.
______________________________________ D, Column Material Density of Voids density of the the fiber, Volume (g/cc) fiber (g/cc) (%) ______________________________________ 0.15 glass* 2.5 94.0 0.15 polyester 1.38 89.1 0.15 polypropylene 0.9 83.3 ______________________________________ *Glass varies in density from about 2.3 to about 2.7 g/cc. The 2.5 g/cc figure used here is in the mid range.
Definition of Pore Diameter
In the definition of various filter media, it will be necessary to use the term "pore diameter". This term as used herein is as determined by the modified OSU F2 test described below.
Wetting of Fibrous Media
When a liquid is brought into contact with the upstream surface of a porous medium and a small pressure differential is applied, flow into and through the porous medium may or may not occur. A condition in which no flow occurs is that in which the liquid does not wet the material of which the porous structure is made.
A series of liquids can be prepared, each with a surface tension of about 3 dynes/cm higher compared with the one preceding. A drop of each may then be placed on a porous surface and observed to determine whether it is absorbed quickly, or remains on the surface. For example, applying this technique to a 0.2 .mu.m porous tetrafluoroethylene (PTFE) filter sheet, instant wetting is observed for a liquid with a surface tension of about 26 dynes/cm. However, the structure remains unwetted when a liquid with a surface tension of about 29 dynes/cm is applied.
Similar behavior is observed for porous media made using other synthetic resins, with the wet-unwet values dependent principally on the surface characteristics of the material from which the porous medium is made, and secondarily, on the pore size characteristics of the porous medium. For example, fibrous polyester, specifically polybutylene terephthalate (hereinafter "PBT") sheets which have pore diameters less than about 20 .mu.m will be wetted by a liquid with a surface tension of about 50 dynes/cm, but will not be wetted by a liquid with a surface tension of about 54 dynes/cm.
In order to characterize this behavior of a porous medium, the term "critical wetting surface tension" (CWST) is defined as follows. The CWST of a porous medium may be determined by individually applying to its surface a series of liquids with surface tensions varying by about 2 to about 4 dynes/cm, and observing the absorption or non-absorption of each liquid. The CWST of a porous medium, in units of dynes/cm, is defined as the mean value of the surface tension of the liquid which is absorbed and that of a liquid of neighboring surface tension which is not absorbed. Thus, in the examples of the two preceding paragraphs, the CWST's are, respectively, about 27.5 and about 52 dynes/cm.
In measuring CWST, a series of standard liquids for testing is prepared with surface tensions varying in a sequential manner by about 2 to about 4 dynes/cm. Ten drops of each of at least two of the sequential surface tension standard liquids are independently placed on representative portions of the porous medium and allowed to stand for 10 minutes. Observation is made after 10 minutes. Wetting is defined as absorption into or obvious wetting of the porous medium by at least nine of the ten drops within 10 minutes. Non-wetting is defined by non-absorption or non-wetting of at least nine of the ten drops in 10 minutes. Testing is continued using liquids of successively higher or lower surface tension, until a pair has been identified, one wetting and one non-wetting, which are the most closely spaced in surface tension. The CWST is then within that range and, for convenience, the average of the two surface tensions is used as a single number to specify the CWST.
A number of alternative methods for contacting porous media with liquids of sequentially varying surface tension can be expected to suggest themselves to a person knowledgeable of physical chemistry after reading the description above. One such involves floating a specimen on the surfaces of liquids of sequentially varying surface tension values, and observing for wet-through of the liquid, or if the fiber used is more dense than water, observing for sinking or floating. Another means would clamp the test specimen in a suitable jig, followed by wetting with the test liquids while applying varying degrees of vacuum to the underside of the specimen.
Appropriate solutions with varying surface tension can be prepared in a variety of ways, however, those used in the development of the product described herein were:
______________________________________ Surface Tension Solution or fluid range, dynes/cm ______________________________________ Sodium hydroxide in water 94-110 Calcium chloride in water 90-94 Sodium nitrate in water 75-87 Pure water 72.4 Acetic acid in water 38-69 Ethanol in water 22-35 n-Hexane 18.4 FC77 (3M Corp.) 15 FC84 (3M Corp.) 13 ______________________________________
Wetting of Fibrous Media by Blood
In packed red cells, as well as in whole blood, the red cells are suspended in blood plasma, which has a surface tension of about 73 dynes/cm. Hence, if packed red cells or whole blood is placed in contact with a porous medium, spontaneous wetting will occur if the porous medium has a CWST of about 73 dynes/cm or higher.
Hematocrit is the percent by volume occupied by red cells. The hematocrit of packed red cells ranges from about 50 to above 80%. Thus, about 50 to over 80% of the volume of PRC consists of the red cells themselves and, for this reason, the surface characteristics of the red cells influence the wetting behavior of PRC. The surface tension has been measured and is given in the literature as 64.5 dynes/cm. ("Measurement of Surface Tensions of Blood Cells & Proteins", by A. W. Neumann et al., from Annals N.Y.A.S., 1983, pp. 276-297.) The lower surface tension of red cells affects the behavior of PRC, for example during priming of filters and during filtration, in ways which are not fully understood.
The benefits conferred by preconditioning fibers to CWST values higher than the natural CWST of PBT and other synthetic fibers include:
Description of the Invention
In accordance with the subject invention, a device and a method for depleting the leucocyte content of a blood product are provided.
The invention comprises a device for the depletion of the leucocyte content of fresh blood products which comprises a fibrous leucocyte adsorption/filtration filter with a pore diameter of from about 0.5 to less than 3.6 .mu.m and having a CWST of from 53 to about 80 dynes/cm.
One of the significant advantages of the device of the invention relates to the priming of the filter assembly (i.e., inducing sufficient flow of PRC to fill the housing), which is more complex and more difficult than would appear at first sight.
If the CWST of the fiber surface is too low, for example that of unmodified synthetic fiber, relatively higher pressure is required to force the PRC to flow through. More seriously, areas of the filter medium tend to remain unwetted, preventing flow of PRC. Further, clotting may occur, especially with finer, high surface area fibers and with older blood.
For reasons which are not well understood, filters which have CWST in excess of about 90 dynes/cm have been observed to have very long priming times, ranging to about 2 to about 5 minutes. It has further been learned, by trial and error, that it is advisable that the CWST be held within a range somewhat above the CWST of untreated polyester fiber (52 dynes/cm), for example, about 55 dynes/cm and higher, and below about 75 or 80 dynes/cm, and more preferably from about 60 to about 70 dynes/cm.
The filter element of the invention has a pore size of from about 0.5 to less than 3.6 .mu.m, preferably from about 0.5 to about 3.5 .mu.m, more preferably from about 0.5 to about 2 .mu.m. This in itself is surprising since such pore sizes are significantly smaller than the size of blood components such as red blood cells which nevertheless pass through. The preferred element is typically made using 2.6 .mu.m average fiber diameter radiation grafted melt blown polybutylene terephthalate (PBT) web, which in a preferred form is hot compressed to a voids volume of about 60% to about 85% and preferably about 65% to about 84% and has a pore diameter of about 0.5 to about 2 .mu.m. The fiber surfaces of the adsorption element are surface grafted to provide a CWST preferably in the range of about 60 to about 70 dynes/cm, such as from about 62 to about 68 dynes/cm. It may be protected from clogging by a gel prefilter and/or by a microaggregate prefilter, and its function is to reduce the leucocyte content by a factor of 30,000 or more while allowing red cells to pass freely.
In a preferred device the fibrous filter medium of the invention is preceded by one or two preformed elements. If a three element filter is used, the function of the first, (the gel prefilter), is to remove gels; that of the second, (the microaggregate filter), is primarily to remove microaggregates though it can also remove some leucocytes by adsorption and by filtration; and the function of the third, the filter medium of the invention (often called hereinafter the adsorption/filtration filter), is to remove leucocytes by adsorption and by filtration. If only two elements are used, the first may be a gel prefilter or a microaggregate filter, followed by the adsorption/filtration filter of the invention. Each of these three elements may comprise one or more separate or integral fibrous layers. The respective elements may differ in their CWSTs, voids volumes, pore sizes, and number of layers. Each element may comprise one or more preforms each containing a number of layers. The respective preforms within each element may also differ with respect to the preceding characteristics.
Significant and novel preferred features of this invention which contribute to achieving high efficiency and capacity for leucocyte removal, and minimize loss of blood within the apparatus include:
Preforming eliminates the pressure on the inlet and outlet faces of the housing which are inherent in a packed fiber system. Preforming also permits one element, for example, the first stage prefilter of the assembled device, to be more or less compressible, yet have a lower or higher density than the one following it. This arrangement contributes to longer life in service.
Preforming makes it more practical to use larger cross sectional area leucocyte depletion devices which have longer life in service, coupled with at least equal and usually better leucocyte removal efficiency, equal or better red cell recovery, and less hold up, when compared with devices that use fibers or fibrous webs packed into a housing at assembly.
Devices have been proposed and some made which comprise various commercially made woven and nonwoven fibrous media as prefilters, along with a more finely pored last stage consisting of non-woven fibrous mats, all packed within a plastic housing. These devices have not had the efficient prefiltration made possible by preforming and, in addition, have been prone to occasional clogging, being too small in cross sectional area.
Because of the difficulty of predicting the consequences of the unusual and variable combination of clogging factors that may be present, even for a person skilled in the art of filter design, it is advisable to incorporate an efficient prefilter.
The present invention, therefore, provides for the optional use of an efficient, small volume gel prefilter system which will contribute to the objective of achieving an average reduction of leucocyte content by a factor of about 30,000 or more, while rarely or never clogging when passing one unit of packed red cells derived from freshly drawn blood.
The use of such an effective gel prefilter which consistently retains at least a substantial proportion of the gel content of one unit of PRC derived from freshly drawn blood is, therefore, a preferred feature of one aspect of this invention. This makes possible the use of a device with a smaller internal volume, with less blood loss due to internal hold-up, while consistently delivering one unit of PRC without clogging.